Dioxins (e.g., 2,3,7,8-Tetrachlorodibenzo-p-dioxin, TCDD) and related compounds (DRCs) are persistent environmental pollutants that gradually accumulate through the food chain, mainly in the fatty tissues of animals. Dioxins are highly toxic and can cause reproductive and developmental problems, damage the immune system, interfere with hormones and also cause cancer. This broad range of toxic and biological effects of DRCs is mostly mediated by the aryl hydrocarbon receptor (AHR).
In animal cells, DRCs bind to AHR in the cytoplasm and then translocate into the nucleus, where they affect the transcription of multiple target genes, including xenobiotic-metabolizing enzymes, such as CYP1A isozymes. AHR is also involved in immune system maintenance, protein degradation and cell proliferation.
The jungle crow (Corvus macrorhynchos) has been considered a suitable indicator for monitoring environmental chemicals such as DRCs. While mammals only have one AHR form, avian species have multiple AHR isoforms such as AHR1 and AHR2. To unveil the functional diversity of multiple avian AHR isoforms in terms of their contribution to responses to DRCs a recent study by Kim et al. investigated the molecular and functional characteristics of jungle crow AHR isoforms, cAHR1 and jcAHR2 (1).
cAHR1 and jcAHR2 proteins were synthesized using AHR proteins were synthesized using the TnT Quick-Coupled Reticulocyte Lysate System to examine whether these jcAHRs have the potential to bind to TCDD. TCDD-binding affinity of the in vitro-expressed jcAHR protein was analyzed using the velocity sedimentation assay with a sucrose gradient.
The results demonstrate that both jcAHR1and jcAHR2 are capable of binding to TCDD.
Long noncoding RNAs have been shown to regulate chromatin states, transcriptional activity and post transcriptional activity (1). Only a few studies have observed long non-coding RNAs modulating the translational process (2). The noncoding RNA BC200 has been shown to inhibit translation by interacting with the translation initiation factors, eIF4A and eIF4B.
To characterize how BC200 translational inhibition could be controlled, a variety of RNAs were transcribed/translated in vitro using the TNT system (Cat. #L4610) from Promega. To each transcription/translation reaction, BC900 RNA, hnRNPE1 and hnRNE2 proteins were added. Inhibition of BC200 activity was noted when proteins were successful expressed (3).
Sosinska, P et.al. (2015) Intraperitoneal invasiveness of ovarian cancer from the cellular and molecular perspective. Ginekol. Pol. 86, 782–86.
The luciferase immunoprecipitation system (LIPS) assay is a liquid phase immunoassay allowing high-throughput serological screening of antigen-specific antibodies. The immunoassay involves quantitating serum antibodies by measuring luminescence emitted by the reporter enzyme Renilla luciferase (Rluc) fused to an antigen of interest. The Rluc-antigen fusion protein is recognized by antigen-specific antibodies, and antigen-antibody complexes are captured by protein A/G beads that recognize the Fc region of the IgG antibody (1).
Researchers and clinicians are fairly certain that all cervical cancers are caused by Human Papillomavirus (HPV) infections, and that HPV16 and HPV18 are responsible for about 70% of all cases. HPV16 and HPV18 have also been shown to cause almost half the vaginal, vulvar, and penile cancers, while about 85% of anal cancers are also caused by HPV16.
E6 is a potent oncogene of HR-HPVs, and its role in progression to malignancy continues to be explored. The E6 oncoprotein of HPV can promote viral DNA replication through several pathways. It forms a complex with human E3-ubiquitin ligase E6-associated protein (E6AP), which can in turn target the p53 tumor-suppressor protein, leading to its ubiquitin-mediated degradation. In particular, E6 from HR-HPVs can block apoptosis, activate telomerase, disrupt cell adhesion, polarity and epithelial differentiation, alter transcription and G-protein signaling, and reduce immune recognition of HPV-infected cells.
Protein-DNA interactions are fundamental processes in gene regulation in a living cells. These interactions affect a wide variety of cellular processes including DNA replication, repair, and recombination. In vivo methods such as chromatin immunoprecipitation (1) and in vitro electrophoretic mobility shift assays (2) have been used for several years in the characterization of protein-DNA interactions. However, these methods lack the throughput required for answering genome-wide questions and do not measure absolute binding affinities. To address these issues a recent publication (3) presented a high-throughput micro fluidic platform for Quantitative Protein Interaction with DNA (QPID). QPID is an microfluidic-based assay that cam perform up to 4096 parallel measurements on a single device.
The basic elements of each experiment includes oligonucleotides that were synthesized and hybridized to a Cy5-labeled primer and extended using Klenow. All transcription factors that were evaluated contained a 3’HIS and 5’ cMyc tag and were expressed in rabbit reticulocyte coupled transcription and translation reaction (TNT® Promega). Expressed proteins are loaded onto to the QIPD device and immobilized. In the DNA binding assay the fluorescent DNA oligonucleotides are incubated with the immobilized transcription factors and fluorescent images taken. To validate this concept the binding of four different transcription factor complexes to 32 oligonucleotides at 32 different concentrations was characterized in a single experiment. In a second application, the binding of ATF1 and ATF3 to 128 different DNA sequences at different concentrations were analyzed on a single device.
Cell-free protein expression is a simplified and accelerated avenue for the transcription and/or translation of a specific protein in a quasi cell environment. An alternative to slower, more cumbersome cell-based methods, cell-free protein expression methods are simple and fast and can overcome toxicity and solubility issues sometimes experienced in traditional E. coli expression systems.
In his webinar, “In vitro, cell-free protein expression–How it helps speed up your research”, Gary Kobs offered an overview of the different cell-free expressions systems offered by Promega and highlighted what needs the different systems best address. He discussed different applications of cell-free expressed proteins and highlighted the combined uses of the HaloTag® Technology with cell-free protein expression. Continue reading “Cell-free Expression: A System for Every Need”
Both prokaryotic and eukaryotic cell-free protein expression systems have found great utility in efforts to screen organic compounds for inhibition of the basic cellular functions of transcription and translation, common targets for antibiotic compounds.
Cell-free systems can provide some advantages over cell-based systems for screening purposes. Cell-free systems allow exact manipulation of compound concentrations. This is an important parameter when evaluating the potential potency of the lead compound.
There is no need for cellular uptake to evaluate the effect of the compounds. While uptake evaluation is important for determining the eventual efficacy of the drug, it can unnecessarily eliminate valuable lead compounds in an initial screen. The interpretation of results in living cells is complicated by the large number of intertwined biochemical pathways and the ever-changing landscape of the growing cell. Cell-free systems allow the dissection of effects in a static system for simpler interpretation of results and the ability to specifically monitor individual processes such as transcription or translation. Individual targets not normally present, or found at low concentrations, can be added in controlled amounts.
The following references illustrate this application:
Cell free protein expression can be utilized for the analysis of: protein/protein interactions, protein nucleic acid interactions, analysis of post translational modifications and many other applications. The majority of these references are based on the characterization of mammalian proteins.
However there are several references using TNT-based systems (either rabbit reticulocyte lysate or wheat germ based) for the analysis of proteins from plants, examples include: Continue reading “Cell-Free Protein Expression: Characterization of Plant Proteins”
The characterization of viral mediated diseases is critical to promote the overall welfare of humans or animals. Initial research focused on the interpretation the genomic content (i.e., DNA or RNA based) of the selected virus. The next step is to better understand the proteins that are encoded by this content and their interaction with the host proteome.
The following citations illustrate the use of cell-free protein expression to facilitate this research. Continue reading “Cell-Free Expression Applications: Characterization of Viral-Mediated Diseases”
As noted in a previous posting the primary use of cell free expression as been the characterization of protein interaction. Due to the convenience of expressing functional protein in a few hours, cell-free expression is also a viable alternative to cell-based expression for other applications. Recent examples include:
Carbonic anhydrase catalyzes the reversible hydration of CO2 and is involved in both C3 and C4 photosynthesis, however its role and intercellular and intracellular locations differ between C3 and C4 plants. Three different cDNA encoding distinct β-carbonic anhydrases were isolated from leaves of the C3 plant Flaveria pringle. To determine if either of the proteins encoded by the cDNA clones are targeted for chloroplasts, [35S]Met labeled proteins were expressed using cell free expression and subjected to chloroplast protein import analysis.
The HspB8-Bag 3 protein complex suppresses mutated Huntington aggregation via autophagy and may involve translational arrest via eIF2 phosphorylation. To evaluate this theory, cell free expression was used to express a clone containing the Huntington exon 1 fragment in the presence of different amounts of recombinant HspB8 protein. To determine if HspB8 induces elF2(αlpha) phosphorylation, recombinant HspB8 was added (in addition to controls) and the extent of phosphor-elF2 levels were determined by Western blotting.
Using In vitro compartmentalization individual genes can be expressed in minute droplets of emulsified cell-free expression system. Formation of the droplet results in co-compartmentalization of the gene and its product, making it possible to select larger gene libraries compared to phage display. This reference illustrates a modification of this technique developed to link genotype and phenotype by fusing the target RNA binding protein to zinc finger proteins that bind to a cognate DNA sequence inserted upstream of the promoter.